Quarky Calcium Release in the HeartNovelty and Significance
Rationale: In cardiac myocytes, “Ca2+ sparks” represent the stereotyped elemental unit of Ca2+ release arising from activation of large arrays of ryanodine receptors (RyRs), whereas “Ca2+ blinks” represent the reciprocal Ca2+ depletion signal produced in the terminal cisterns of the junctional sarcoplasmic reticulum. Emerging evidence, however, suggests possible substructures in local Ca2+ release events.
Objective: With improved detection ability and sensitivity provided by simultaneous spark–blink pair measurements, we investigated possible release events that are smaller than sparks and their interplay with regular sparks.
Methods and Results: We directly visualized small solitary release events amid noise: spontaneous Ca2+ quark-like or “quarky” Ca2+ release (QCR) events in rabbit ventricular myocytes. Because the frequency of QCR events in paced myocytes is much higher than the frequency of Ca2+ sparks, the total Ca2+ leak attributable to the small QCR events is approximately equal to that of the spontaneous Ca2+ sparks. Furthermore, the Ca2+ release underlying a spark consists of an initial high-flux stereotypical release component and a low-flux highly variable QCR component. The QCR part of the spark, but not the initial release, is sensitive to cytosolic Ca2+ buffering by EGTA, suggesting that the QCR component is attributable to a Ca2+-induced Ca2+ release mechanism. Experimental evidence, together with modeling, suggests that QCR events may depend on the opening of rogue RyR2s (or small cluster of RyR2s).
Conclusions: QCR events play an important role in shaping elemental Ca2+ release characteristics and the nonspark QCR events contribute to “invisible” Ca2+ leak in health and disease.
The Ca2+ ion is the most versatile intracellular messenger involved in vital cellular processes that include contraction, secretion, apoptosis, and gene expression regulation underlying cell proliferation and differentiation.1 In cardiac cells, Ca2+ entry through voltage-gated Ca2+ channels in the plasma membrane during an action potential triggers intracellular Ca2+ release through the type 2 ryanodine receptor (RyR2) Ca2+ release channels in the sarcoplasmic reticulum (SR), a process known as Ca2+-induced Ca2+ release (CICR). Depending on species and developmental stage, the SR amplification of the Ca2+ influx varies from almost 0 (early development) up to 16 (adult rat or mouse heart).2,3
As one continuous organelle,4 the SR takes the form of an elaborated nanoscopic tubular and cisternal network that extends throughout the entire cytoplasm but occupies only ≈4% of the “cytoplasmic” volume in rabbit ventricular myocytes.5 It is also connected to both the endoplasmic reticulum and the nuclear envelope Ca2+ stores that are implicated in noncontractile Ca2+ signaling.4 Ca2+ release from the cardiac SR is controlled almost exclusively by RyR2 Ca2+ release channels. The SR membrane harbors ≈106 RyR2s per myocyte, and ≈30 to 300 RyR2s form a 2D paracrystalline array or Ca2+ release unit (CRU). A 3D constellation of ≈104 CRUs are spangled regularly throughout the cell.6,7 Most recent data suggest that some RyR2s within the regular organization of an array could be missing,8,9 suggesting that clusters of RyR2s of different sizes (including rogue RyR2s10) could share the same junctional SR (jSR).
The prevalent view is that activation of a single CRU gives rise to a local, discrete Ca2+ release event: a Ca2+ spark.11 Summation of thousands of Ca2+ sparks that are activated during each heart beat gives rise to a whole-cell [Ca2+]i transient during cardiac excitation–contraction coupling.12,13 Simply put, this view suggests that there are no other forms of Ca2+ release but sparks in cardiac myocytes under physiological conditions. Several lines of observation, however, have challenged this view. Lipp and Niggli initially reported that CICR-dependent SR Ca2+ release may occur in a spatially uniform fashion under specific conditions.14 They ascribed the unresolved fundamental Ca2+ release events to the subresolution entity dubbed “Ca2+ quarks.” Later, they supported this hypothesis by showing that 2-photon photolysis of Ca2+-caged compounds can trigger local Ca2+ release events that appears to be smaller than sparks.15 Using loose-seal patch-clamp and confocal imaging techniques, Cheng and colleagues demonstrated that individual release events evoked from the same CRU differ significantly by virtue of amplitude, suggesting polymorphism of Ca2+ sparks.16 Moreover, Sobie et al hypothesized that rogue RyR2s may act to produce SR Ca2+ efflux at a lower level than seen with the stereotyped CRU activation that produces Ca2+ sparks.10 Collectively, these reports question whether or not Ca2+ sparks are the sole “elementary” event underlying SR Ca2+ release. Alternatively, Ca2+ sparks may possess intriguing characteristics that are still unknown to us despite a 15-year intensive investigation.
As a Ca2+ spark appears, a local SR Ca2+ depletion signal, called a Ca2+ blink, can be observed.5 Blinks and sparks are 2 manifestations of the same elementary Ca2+ release event, viewed from the SR lumen and the cytosolic space, respectively. The SR consists of special regions that include the jSR and the free SR (fSR), and each component is distinct with varying concentrations of lumenal Ca2+ buffers (eg, calsequestrin, calreticulin), membrane-linked channels and transporters (eg, RyR2s, SR Ca2+ ATPase [SERCA]2a) and regulatory proteins (eg, triadin, junctin, phospholamban). The SR lumen does not appear to include cytosolic Ca2+ regulatory and buffering proteins (eg, troponin, calmodulin). As such, characterization of Ca2+ blinks, particularly when done in parallel with Ca2+ sparks, should be able to provide unique insight into SR Ca2+ release mechanisms and, more globally, into intracellular Ca2+ signaling.
In the present study, we exploit Ca2+ spark–blink pairs to investigate local Ca2+ release events with improved detection ability and sensitivity. Our results reveal that low-flux quarky Ca2+ release (QCR) events coexist and interplay with regular Ca2+ sparks to provide rich Ca2+ signaling substructure and complexity (eg, dictating the decay of the sparks and the restoration of the blinks). This QCR component appears to be coupled to the high-flux initial release in a spark via the CICR mechanism and may have arisen from activation of rogue RyR2s or complex regulation of RyR2s within an array. Our results also show that the Ca2+ leak mediated through the QCR mechanism appears to be at least as important as that through the Ca2+ spark mechanism.
To simultaneously visualize Ca2+ sparks and Ca2+ blinks, New Zealand White rabbit ventricular myocytes were first incubated for 2 hours at 37°C with the low-affinity Ca2+ indicator fluo-5N-AM (20 μmol/L, Invitrogen) and then for 15 minutes at room temperature with the high-affinity Ca2+ indicator rhod-2-AM (5 μmol/L, Invitrogen).5 With the diastolic spark frequency being very low in rabbit ventricular myocytes, the SR Ca2+ load was increased by replacing extracellular Na+ (NaCl) by equimolar Li+ (LiCl) in the bath solution, unless otherwise noted (see Figures 3 and 5).
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Spark–Blink Pair Analysis Increased Detection Ability of In-Focus Ca2+ Release Events
Figure 1A showed a representative Ca2+ spark–blink pair in line-scan images from a rabbit ventricular myocyte. Spatial profiles (Figure 1B) revealed that the blink aligned well with the jSR (J) and was sharply confined (full-width at half maximum [FWHM]=1.01±0.05 μm; n=51), reflecting the nanometer-sized jSR ultrastructure and restricted intra-SR Ca2+ diffusion between the jSR and the fSR.5 In contrast, the companion spark, which reflected the local Ca2+ release, Ca2+ diffusion, and buffering in the cytosolic space, spanned the entire sarcomere and even spread into the neighboring junctions (J-1, J +1), displaying a FWHM of 2.27±0.12 μm (n=51) and a volume 10 times greater than for a blink. Because of the sharp spatial delineation of Ca2+ blinks, we have exploited blinks as the guide to select in-focus release events, ie, those sparks associated with discernible blinks. Under the present experimental conditions, ≈60% of Ca2+ sparks were rejected as out-of-focus events for the lack of companion blinks.
Simultaneous measurement of Ca2+ sparks and Ca2+ blinks provided us with the unprecedented ability to identify small local Ca2+ release fluxes amid background noise. Using Gaussian noise–simulated traces, we showed that dual channel simultaneous detection suppresses false-positive events by ≈150-fold as compared with single-channel detection (Online Figure I), allowing the detection of a population of QCR events (see below).
Quarky Ca2+ Release: Novel Type of Local Ca2+ Signaling
Figure 2A and 2B shows an example of a succession of spark–blink pairs and QCR–quarky Ca2+ depletion (QCD) pairs on the same jSR. The kinetics of these events were very short (QCR: tpeak=19.1±1.0 ms, t67=20.1±1.1 ms; QCD: tnadir=19.2±1.3 ms, t67=20.8±1.9 ms; n=42; Figure 2C). The amplitude of these reciprocal QCR-QCD events were only one-tenth to one-third of the regular spark–blink events (QCR: ΔF/F0=0.069±0.006; QCD: ΔF/F0=0.025±0.002; n=42; Figure 2D), suggesting that the QCR events may arise from a mode of Ca2+ release that is different from that which underlies the regular spark–blink pairs. This idea was substantiated by the amplitude histogram that suggests the existence of 2 distinct populations of Ca2+ release events (Figure 2E; n=176). Nevertheless, the properties of QCR events still appear larger than the isolated opening of a single RyR2 (ie, the true Ca2+ “quarks” hypothesized by Lipp and Niggli14) because the Ca2+ flux of a single RyR2 Ca2+ quark is expected to be even smaller with a duration much shorter because of rapid closing by stochastic attrition.17
To evaluate the significance of these QCR events in more physiological conditions, we studied QCR when rabbit ventricular myocytes were electrically stimulated (0.5 Hz) in a regular HEPES buffer (Figure 3A). We found that QCR events were 10 times more frequent than spontaneous Ca2+ sparks (2.77±0.31 versus 0.24±0.06 [100 μm]−1sec−1; Figure 3B), whereas sparks displayed a 10 times greater signal mass (7.47±1.55 versus 0.62±0.13; Figure 3C). Consequently, the summed SR Ca2+ leak attributable to sparks was similar to that mediated by QCR events (1.48±0.37 versus 1.49±0.34). Taking into account that numerous QCR events may have gone undetected, we conclude that QCR represent an important, heretofore underappreciated SR Ca2+ leak mechanism (see below).
Spark–Blink Kinetics: Evidence for Low-Flux Continued Ca2+ Release
Careful examination of the kinetics of Ca2+ spark–blink pairs revealed substantial variation with respect to the decay of the sparks and the restoration of the blinks (Figure 4). Specifically, the spark decay time (t67) exhibited a broad distribution with a mode at 45 ms, ranging from 25 ms (at 5% cutoff of the lower limit) to 95 ms (at 95% cutoff of the upper limit; Figure 4A). Such a large variation was unexpected (see Discussion) but could not be dismissed as an artifact of confocal detection because we observed a similar large variation in blink recovery time (t67) (Figure 4A), the latter being essentially undistorted by confocal sampling.5
To investigate whether this variability could simply be attributed to regional differences in Ca2+ diffusion or buffering, we examined the relationship between Ca2+ sparks and their corresponding Ca2+ blinks, the latter reflecting local jSR depletion and refilling. Because Ca2+ diffusion and buffering differ markedly in the SR lumen versus the cytosol,18 we had expected little correlation between the recovery of blinks and the decay of their companion sparks. However, both Ca2+ sparks and blinks exhibited similar average durations (tpeak=30.4±1.3 ms and t67=54.3±2.8 ms for sparks versus tnadir=29.6±1.4 ms and t67=55.5±2.6 ms for blinks, n=51 from 31 cells). In addition, scatter plots showed a strong linear correlation between the t67 of spark decay and the t67 of blink recovery, with a slope of 0.88 and r2=0.91 (Figure 4B).
To further study the kinetics of the spark–blink pairs, we grouped spark–blink pairs based on t67 of the sparks, which usually display better signal-to-noise ratios than the blinks. For events in the same group (t67<50 ms, n=24 events; 50 ms<t67<70 ms, n=19 events; or t67>70 ms, n=7 events), we aligned the simultaneously recorded sparks and blinks using the spark peak as the reference point, and resultant averaged traces are shown in Figure 4C. The longer the spark decay, the slower the corresponding blink recovery. More strikingly, once normalized by the amplitude, sparks and blinks showed virtually overlapped time courses in every group (Figure 4C). The tight correlation between spark and blink kinetics indicates that a common mechanism underlies the variability both in the cytosol and the SR lumen. That the average t67 value varied by more than 2-fold in the 3 groups suggests that this variability is not readily attributable to a change in local Ca2+ recycling by the SERCA2a, which plays only a minor role in shaping the spark kinetics.19 Rather, these results indicate the presence of low-flux continued Ca2+ release that dictates the decay-recovery kinetics of the spark–blink pairs.
In contrast, the initial Ca2+ release appears to be stereotyped: there was little variation in the time-to-peak of the spark or time-to-nadir of the blink. The average value (≈30 ms) was similar for all different categories of spark–blink pairs (Figure 4D); sparks or blinks displayed similar amplitudes regardless of their duration (Figure 4E). Moreover, there was little correlation between the amplitude and t67 of sparks (r2=0.0028; Figure 4F). This result indicates that, once triggered, the continued release component is self-sustained, independently of the size of initial release. Collectively, our data support the notion that the spark–blink pair consists of a stereotyped initial release component and a highly variable small flux trailing release component.
Effects of EGTA on Continued Ca2+ Release
To test the hypothesis that the trailing small-flux release was triggered by initial high flux release via local CICR, we examined Ca2+ spark–blink kinetics in the presence of 2 EGTA concentrations (0.5 or 2 mmol/L) in chemically permeabilized myocytes (Figure 5A and 5B). As an exogenous Ca2+ chelator, EGTA can effectively reduce the physical size and duration of Ca2+ sparks in the cytosol.20 However, EGTA was expected to exert no such effect on the companion Ca2+ blinks as long as Ca2+ release process remained unaltered. Should EGTA enter the SR, it should not materially affect the dynamics, either, because its high affinity for Ca2+ (Kd≈150 nmol/L at pH 7.2) renders it saturated at the high levels of [Ca2+]SR normally observed (in the range of a few hundreds micromolar to a few millimolar).
We found that in the presence of 0.5 mmol/L EGTA, Ca2+ spark and Ca2+ blink t67 values were 40.6±2.2 and 44.7±2.4 ms (n=58), respectively, which were 13.7 and 10.8 ms faster than the spark–blink pairs in intact cells in the absence of EGTA. Furthermore, in 2 mmol/L EGTA, Ca2+ spark and Ca2+ blink t67 values were further shortened to 29.1±2.4 and 31.7±2.5 ms (n=26, P<0.005), respectively (Figure 5C). Although the EGTA shortening of spark duration has been demonstrated before, the EGTA effect on Ca2+ blinks was totally unexpected for the reasons discussed above. The shortening of Ca2+ blinks suggested that the Ca2+ release process itself has been abbreviated by the inclusion of the Ca2+ buffer in the cytosol. Such EGTA sensitivity provided the first experimental evidence that a CICR-dependent mechanism participates in the release of an ongoing spark.
Analysis of the signal amplitudes showed that the Ca2+ spark amplitude decreased from 0.96±0.06 (n=58) in 0.5 mmol/L EGTA to 0.65±0.06 (n=26, P<0.01) in 2 mmol/L EGTA, as expected. By contrast, the blink amplitude displayed no significant change (0.22±0.01, n=58 in 0.5 mmol/L EGTA versus 0.20±0.01, n=26 in 2 mmol/L EGTA, P>0.05; Figure 5E), suggesting that the amount of the initial SR Ca2+release was essentially unchanged. The time-to-nadir of the blink was only marginally shortened (26.6±0.81, n=58, and 23.5±0.95 ms, n=26, in 0.5 and 2 mmol/L EGTA, respectively, P<0.01; Figure 5C), indicating that the initial release, which determined the peak and nadir of the spark–blink pair, was largely EGTA-insensitive. Regardless of EGTA concentration, there was a linear correlation between spark and blink t67 values (Figure 5D), as was the case in intact cells in the absence of EGTA (Figure 4B). Taken together, these results reinforce the idea that elemental SR Ca2+ release events consist of a large EGTA-resistant initial release flux followed by a trailing EGTA-sensitive lower-flux release of variable duration.
Substructures in Prolonged Spark–Blink Pairs
The aforementioned data suggested that Ca2+ release continues beyond the peak or the nadir of the spark–blink pair, which confers the variability to the decay-recovery kinetics. We further hypothesized that such continued low-flux Ca2+ release is of the same or similar nature as the solitary QCR events described above. Two questions arise from these observations: (1) How can we directly visualize QCR-QCD substructures in a spark–blink pair? (2) How does the presence of the QCR mechanism fit with robust Ca2+ spark (and Ca2+ blink) termination? In this regard, we noticed a subpopulation of prolonged Ca2+ sparks (t67>mean+4 SD=135 ms) occurring spontaneously in intact rabbit ventricular myocytes, examples of which are shown in Figure 6.
Figure 6A shows a spontaneous long-lasting Ca2+ spark that rises to a peak and then develops a low long-lasting plateau before its termination, similar to the prolonged sparks induced by FK506, rapamycin, or low-dose ryanodine in rat ventricular myocytes.11,17 The companion blink displays a clear nadir at the peak of the spark and then returns toward a low plateau. The sustained low [Ca2+]SR signal is consistent with continued Ca2+ efflux and reflects a dynamic balance between the efflux and the refilling of the jSR. Presumably, the prolonged or sustained Ca2+ sparks and blinks arise from sustained local CICR at a single jSR attributable to one or more RyR2s remaining active/open, a suggestion consistent with the low-dose ryanodine (or other) treatment. More complex examples of sustained release are shown in Figure 6B and 6C, illustrating repetitive activation of the QCR mechanism during prolonged spark–blink pairs. In both examples, the kinetics of the [Ca2+]SR closely mirrored the kinetics of the sparks. The succession of “bumps” (QCR events) of the prolonged Ca2+ spark are matched by “dips” (QCD events) of the prolonged Ca2+ blink, with an overall negative correlation coefficient r=−0.89 and −0.87, respectively. Interestingly, the long spark–blink pairs were not seen in 2 mmol/L EGTA (Figure 5), consistent with CICR triggering of QCR-QCD events during prolonged Ca2+ sparks.
Novel features of Ca2+ release from the SR in heart cells have been identified and characterized here by dual imaging of Ca2+ signals in the cytosol and in the lumen of the SR. In addition to Ca2+ sparks, we report on the detection and characterization of small amplitude local Ca2+ release events that are spatially and temporally well-confined (ie, QCR-QCD events). The Ca2+ leak mediated by these QCR events was at least as important as those by Ca2+ sparks. We also show that similar low-flux Ca2+ release events can occur in conjunction with a regular Ca2+ spark. The latter is vividly manifested by the trailing QCR-QCD events in a prolonged Ca2+ spark and may underlie the variability in the decay kinetics of a Ca2+ spark. The newly identified QCR and substructure of Ca2+ signals in a spark (see Figure 7) provoke a number of questions that are discussed below.
Local Ca2+ Release During a Spark
Although polymorphism of Ca2+ spark amplitude has been demonstrated previously,16,21 here, we show for the first time that the kinetics of in-focus Ca2+ sparks display duration variation ascribed to the variability of the declining phase. Surprisingly, there is a tight correlation between kinetics of sparks and their corresponding blinks despite marked differences in calcium diffusion and buffering in the cytosol and the SR lumen. Moreover, increased cytosolic Ca2+ buffering by EGTA similarly abbreviates both Ca2+ sparks and Ca2+ blinks. These results suggest a number of important features of the spark release function. First, the SR Ca2+ release flux that contributes to the Ca2+ spark must last longer than initially hypothesized,11 ie, it must continue after the peak of the spark. Second, the release fluxes that underlie Ca2+ sparks appear to be a decreasing function of time, as also suggested by derivation of the release function from spark measurement22 and in the modeling work of Sobie et al.17 Third, the SR Ca2+ flux is long enough and strong enough to dominate the effect of cytosolic Ca2+ diffusion on spark kinetics, and the effect of jSR refilling on the blink kinetics. These findings in cardiac cells are also in general agreement with reports on “Ca2+ embers” under various experimental conditions in skeletal muscles,23 although, unlike heart, square-wave release fluxes are thought to be the norm.24 It should also be noted that Zima et al25 recently reported a very long blink recovery time (≈161 ms on average) that was attributed to a slow intra-SR Ca2+ diffusion caused by restrained connection between fSR and some jSR. However, our data suggest that the recovery of blinks can be very fast, as evidenced by the population of blinks with t67<70 ms in the absence and presence of millimolar EGTA. In light of the present findings, a more plausible explanation for the findings of Zima et al26 is that Ca2+ sparks can have variable kinetics and can display enhanced trailing SR Ca2+ release, which would confer the unexpected slow recovery of the associated Ca2+ blink.
Furthermore, we show that the initial high Ca2+ release flux in a Ca2+ spark is stereotypical, in contrast to the trailing Ca2+ release flux. More importantly, the trailing, but not the initial, Ca2+ release flux is sensitive to cytosolic Ca2+ buffering by EGTA. Hence, the local SR Ca2+ release during a Ca2+ spark occurs in 2 modes (one that involves a high-flux Ca2+ release that operates in a largely all-or-none fashion; and a second that involves low-flux Ca2+ release that is highly variable). The fact that EGTA abbreviates the spark and blink duration by selectively acting on the low-flux release mode is important because it indicates that the trailing release is activated and sustained by a CICR mechanism and may be more susceptible to modulation or perturbation by physiological and pathophysiological factors, as well as changes in experimental conditions.
Possible Mechanism Underlying Subtler Local Ca2+ Release
Although QCR can occur at the same site as regular sparks (at the optical resolution), QCR events have distinct features including their small size and apparent lack of refractoriness; the latter is evidenced by repetitive activation of QCR events in long sparks. Our working hypothesis (Figure 7) is based on recent publications from the Soeller8 and Hoshijima9 laboratories. Using optical superresolution technique (PALM)8 or electron tomography,9 they have shown that large clusters of RyR2s constituting the CRU were incompletely filled with RyR2. This leaves the possibility for some RyR2s to be rogue (single or in a small cluster) and in close proximity to the large cluster of RyR2s. Such rogue RyR2s may display higher CICR sensitivity (at low [Ca2+]i levels) than the large cluster of RyR2s.10 Using our previous numeric spark model,26 the differential effect of EGTA (between 0.5 and 2 mmol/L) on spark kinetics was modest because t67 was only reduced by 5.5 ms in the presence of 2 mmol/L EGTA. In contrast, our experimental data showed that 2 mmol/L EGTA reduced t67 by 11.5 ms. If we consider an opening duration of rogue RyR2s of 1 ms27 and a release current of 5% of the initial release of a Ca2+ spark, the opening of 6 successive rogue RyR2s 10 ms after the peak of the spark would increase t67 by ≈8 ms. The question whether or not these rogue RyR2s would be on the same jSR than larger clusters of RyR2s has also been further answered by the EGTA experiments. Our modeling suggested that 2 mmol/L EGTA can indeed exert substantial buffering effect over the distance of a half or full CRU (average width of 465 nm5; Online Figure II).
In our Ca2+ spark and QCR model, solitary QCR-QCD events arise from spontaneous activation of rogue RyR2s (Figure 7B), whereas the spark-commingled QCR events are activated by preceding CRU release via the CICR mechanism (Figure 7A). An important distinction of CRU and rogue release is that the former has a steeper [Ca2+]i dependence attributable to the cooperativity of interconnected RyR2s.10 This logic suggests 2 additional features. First, QCR-QCD events from rogue RyR2s would rarely trigger their nearby “master” CRU (in a retrograde manner). Second, repetitive openings of rogue RyR2 U (or CICR interplay among a few rogue RyR2 U) explain the high variability and the EGTA sensitivity of the trailing release. As a cautionary note, we have assumed that isolated QCR and the straggling QCR on the tail of long sparks are of common mechanism, as judged by their phenotypic similarities. Nevertheless, the similarities may be only apparent, and they may arise from distinct populations of rogue RyR2s. Future investigations are warranted to discriminate these possibilities.
Implications in SR Ca2+ Leak
All means by which Ca2+ exits the SR is “Ca2+ leak.” The most common measurement of Ca2+ leak is Ca2+ spark. Larger SR Ca2+ releases like macrosparks, long sparks, or Ca2+ waves are also easy to detect. However, so far, the small SR Ca2+ release events have been elusive until now, with the new results described here. By using spark–blink pairs as a guide to identify events amid noise, we have been able to show that QCR is a new form of active Ca2+ leak that otherwise would have been “invisible” or dismissed as nonsignificant. Because the real frequency of QCR events is difficult to estimate, we can only suggest that the real contribution of these small release events to Ca2+ leak is at least as important as Ca2+ leak caused by sparks. In addition, when a QCR is occurring, the nearby jSR could be more likely to have a spontaneous Ca2+ spark or participate in a conducted wave. Overall, this component of Ca2+ leak may be part of an invisible Ca2+ leak that might become aggravated in disease. The existence of this invisible leak may also be of importance in setting the integrated “pump–leak balance” of the SR and thereby in the fine tuning of local and global Ca2+ signaling.
In summary, we have demonstrated substructures in elementary SR Ca2+ release events and identified a new population of subtler release events, the QCR, which can be seen when the viewing is guided by “spark–blink pair” measurements. The cumulative Ca2+ leak associated with QCR appears to be as large as those associated with spontaneous Ca2+ sparks. Evidence suggests that the QCR may depend on the opening of rogue RyR2s or complex CRU regulation of RyR2s. Taken together, the understanding of coexistence and interplay of quarky and spark Ca2+ release events and their distinct regulation leads us to novel and important insight into cardiac Ca2+ signaling. If supported by additional independent evidence, our new understanding of QCR may lead to novel strategies for the control of SR Ca2+ leak and for the treatment of heart failure and Ca2+-dependent arrhythmias.
Sources of Funding
This work was supported in part by the Intramural Research Program of the NIH, National Institute on Aging (to D.Y.); National Heart, Lung, and Blood Institute grants (grants P01 HL67849 and R01-HL36974), Leducq North American-European Atrial Fibrillation Research Alliance, European Union Seventh Framework Program (FP7) “Identification and therapeutic targeting of common arrhythmia trigger mechanisms” by European Community's Seventh Framework Programme FP7/2007–2013 under grant agreement HEALTH-F2-2009-241526, EUTrigTreat and support from the Maryland Stem Cell Commission (to W.J.L.); and the Chinese Natural Science Foundation (30630021) and the Major State Basic Science Development Program (2007CB512100, 2011CB809100) (to H.C.).
In October 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.9 days.
Non-standard Abbreviations and Acronyms
- Ca2+-induced Ca2+ release
- Ca2+ release unit
- free sarcoplasmic reticulum
- junctional sarcoplasmic reticulum
- quarky Ca2+ depletion
- quarky Ca2+ release
- type 2 ryanodine receptor
- sarcoplasmic reticulum Ca2+ ATPase
- sarcoplasmic reticulum
- Received June 4, 2009.
- Revision received January 17, 2010.
- Revision received November 17, 2010.
- Accepted November 30, 2010.
- © 2011 American Heart Association, Inc.
- Escobar AL,
- Ribeiro-Costa R,
- Villalba-Galea C,
- Zoghbi ME,
- Perez CG,
- Mejia-Alvarez R
- Wu X,
- Bers DM
- Brochet DX,
- Yang D,
- Di Maio A
- Soeller C,
- Crossman D,
- Gilbert R,
- Cannell MB
- Baddeley D,
- Jayasinghe ID,
- Lam L,
- Rossberger S,
- Cannell MB,
- Soeller C
- Hayashi T,
- Martone ME,
- Yu Z,
- Thor A,
- Doi M,
- Holst MJ,
- Ellisman MH,
- Hoshijima M
- Cheng H,
- Lederer WJ,
- Cannell MB
- Cannell MB,
- Cheng H,
- Lederer WJ
- Lopez-Lopez JR,
- Shacklock PS,
- Balke CW,
- Wier WG
- Wang SQ,
- Stern MD,
- Rios E,
- Cheng H
- Gonzalez A,
- Kirsch WG,
- Shirokova N,
- Pizarro G,
- Stern MD,
- Rios E
- Zima AV,
- Picht E,
- Bers DM,
- Blatter LA
- Zahradnikova A,
- Zahradnik I,
- Gyorke I,
- Gyorke S
Novelty and Significance
What Is Known?
Ca2+ sparks represent the elemental units of Ca2+ release from the sarcoplasmic reticulum (SR) of the cardiac myocyte.
Ca2+ depletion from the junctional (j)SR during a Ca2+ spark can be measured and has been termed a “Ca2+ blink.”
Clusters of type 2 ryanodine receptors (RyR2s) can be different sizes but could share the same jSR.
What New Information Does This Article Contribute?
Imaging jSR and cytosolic Ca2+ simultaneously enables the detection of subtle Ca2+ release events that otherwise would be difficult to discriminate from noise.
Using this method, we detected low amplitude, solitary Ca2+ release events, referred to as quarky Ca2+ release (QCR), which occurred either independently or during the declining phase of a full amplitude Ca2+ spark.
QCR events, but not the primary spark-mediated Ca2+ release, were suppressed by the slow Ca2+ buffer EGTA, indicating that they were triggered by a Ca2+-induced Ca2+ release (CICR) mechanism. The stochastic recruitment of QCR events spawned by the Ca2+ spark plausibly explains the variability of spark duration.
In paced myocytes, QCR events were so frequent that the SR Ca2+ leak from these events could be equal to that through Ca2+ sparks.
QCR events not associated Ca2+ sparks could contribute to “invisible” Ca2+ leak in health and disease.
Ca2+ sparks represent the elemental unit of Ca2+ release. They result from the activation of large arrays of RyR2s. Recently, it has been shown that clusters of RyR2s of different sizes could share the same jSR, suggesting possible substructures in local Ca2+ release events. We tested this hypothesis by visualizing QCR events, which probably depend on the opening of small clusters of RyR2s (or rogue RyR2s). QCR events might also be responsible for the variability of the declining phase of a spark. Because the frequency of QCR events in paced myocytes is much higher than the frequency of Ca2+ sparks, the total Ca2+ leak caused by small QCR events is approximately equal to that of the spontaneous Ca2+ sparks. This new mode of Ca2+ release may be the mechanism behind the so-called “invisible” Ca2+ leak. These findings suggest new approaches for the treatment of heart diseases characterized by exaggerated SR Ca2+ leak, such as heart failure and catecholaminergic polymorphic ventricular tachycardia.